Part:BBa_K3033019

BBa_K3033019: OPHTII Capture system version 2: eGFP tagged at the N-terminal 2-Tyrosine capturing d

Overview

Our first and main feature of the whole Self-Activating Nanoparticle E.coli organism (SANCE). The Surface Nanoparticle Capturing Protein (SNCP). This variant name of this SNCP is OPHTII Capture system version 2: eGFP tagged at the N-terminal 2-Tyrosine capturing domain The general overview of the whole feature is shown in Figure 1 below.

Figure 1: Overview of the mechanism and where our SNCP translocates in our bacteria

Our surface nanoparticle capturing protein (SNCP) has a couple of components as shown in figure 2. An anchoring protein. A linker enzyme. Next is the presence of 6-histidine tag. Finally, the capturing domain. Our inspiration for this capturing system is from the Mefp-5 protein that is found on the foot of the mussels. This inspiration protein has L-Dopa and histidine repeats in them thus we want to express L-Dopa as our capturing domain as it has a sticky property.

Figure 2: Overview of the mechanism and where our SNCP translocates in our bacteria

Our system would primarily be anchored onto the cell membrane of the bacteria using the outer membrane protein called OmpW derived from E. coli itself, it forms an 8-stranded beta-barrel with a long and narrow hydrophobic channel. We chose this as our anchoring protein as it is very stable and is able to carry our other protein onto the surface of the bacteria. We have truncated this version of OmpW at the 191st amino acid as it has been shown to be stably anchored onto the cell membrane while being able expose the rest of the proteins onto the outside of the cell membrane. The next component is the Perhydrolase which was derived from the organism Pseudomonas aeruginosa. According to our adviser, this would be enhancing our capturing system’s capability thorough the process of epoxidation. The next component is the 6- histidine tag which would be used to check the protein expression.

Protein Expression tests

Figure 3: This western blotting result shows the band signals for all the variants for our surface nanoparticle capturing protein (SNCP)

Figure 4: Immunofluorescence imaging result of our bacteria transformed with our Surface nanoparticle capturing protein (SNCP)

We want to examine whether our target protein is being expressed in our 5 different surface NPs capturing protein: OPHT1, OPHT2, OPHT3, OPHM, and OPHA. From Figure A-9, we can observe that our OPHT1, OPHT2, and OPHA show the band on the correct size. OPHT1 and OPHA1 show bands at around 50kDa, while OPHT2 which tagged with GFP shows the band at around 75kDa; which are at the estimated protein band size. However, unfortunately, OPHT3 and OPHM did not show any protein band. Although there are no positive results from western blot in OPHT3 and OPHM, the green light from the anti-mouse secondary antibody can be seen in immunofluorescence (IF) assay which shows that the all adhesion constructive system express on the surface of our bacteria.

Functional Tests

Yellow fluorescent nanospheres from Spherotech catalog number FP-00552-2 with and excitation wavelength of 440 nm and emission wavelength of 480 nm; with the size of 40 nm to 90 nm were used in all of the test.

Figure 4: RFU of 5 variants and wild-type bacteria after 6-minute treatment and RFU of 5 variants and wild-type bacteria after 12-minute treatment

Figure 5: RFU of 5 variants and wild-type bacteria after 24 minute treatment and RFU of 5 variants and wild-type bacteria after 48 minute treatment

Based on the successful capturing protein expression results, we want to test the nanoparticle binding capability for future potential application. We incubated our construct variant bacteria, which are expressing NPs capturing protein, with nanoparticles and measured the fluorescence signal to quantify NPs (nanospheres with fluorescence) signals in the supernatant after centrifugation. The fluorescence delta change is determined by individually subtracting the absorbance readings of the negative control and construct variants, which both incubated with nanoparticle, to the positive control reading, which contains the same type and concentration of nanoparticle in phosphate buffer. The greater the delta change value is, the more efficient our capturing protein function is. In conclusion, OPHM and OPHT have consistently high Relative Fluorescence Unit (RFU) throughout 6, 12, 24 and 48 minutes incubation time from Figure A-11 to A-14. In terms of incubation time, 6 minutes is considered as the best incubation time length for bacteria to capture the most amount of nanoparticles from Figure A-11. The figures above show that compared to the wild-type bacteria, consistent in different time points, our bacteria with the surface nanoparticle capturing protein performs better.

Figure 6: Contrast of nanoparticle concentration (ppm) between in pellet and in supernatant collected from 5 construct variants with different incubation time length

These declining trendlines show that the observed increase in the concentration of nanoparticles in the pellet and is reflected clearly as a decrease of the nanoparticle concentration in the supernatant as shown as in Figure 6. This shows that our Surface nanoparticle capturing protein is working as intended.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 2234
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]